Clinical validation and implementation of droplet digital PCR for the detection of BRAF mutations from cell-free DNA
Details
Publication Year 2022-10,Volume 54,Issue #6,Page 772-778
Journal Title
Pathology
Publication Type
Research article
Abstract
Droplet digital PCR (ddPCR) has been demonstrated in many research studies to be a sensitive method in the analysis of circulating tumour DNA (ctDNA) for identifying mutations and tracking disease. The transition of ddPCR into the diagnostic setting requires a number of critical steps including the assessment of accuracy and precision and ultimately implementation into clinical use. Here we present the clinical validation of ddPCR for the detection of BRAF mutations (V600E and V600K) from plasma. We describe the performance characteristics assessed including the limit of blank, limit of detection, ruggedness, accuracy, precision and the effect of the matrix. Overall, each assay could achieve a limit of detection of 0.5% variant allele fraction and was highly accurate, with 100% concordance of results obtained from routine diagnostic testing of formalin fixed tumour samples or reference controls (n=36 for BRAF V600E and n=30 for BRAF V600K). Inter-laboratory reproducibility across 12 plasma samples for each assay was also assessed and results were 100% concordant. Overall, we report the successful validation and translation of a ddPCR assay into clinical routine practice.
Keywords
*Cell-Free Nucleic Acids; *Circulating Tumor DNA/genetics; DNA Mutational Analysis/methods; Formaldehyde; Humans; Mutation; Polymerase Chain Reaction/methods; Proto-Oncogene Proteins B-raf/genetics; Reproducibility of Results; Braf; Cell-free DNA; droplet digital PCR; melanoma
Department(s)
Pathology; Laboratory Research; Medical Oncology
PubMed ID
35618509
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